1. Field of the Invention
This invention relates to a rapid, convenient process for distinguishing target nucleic acid segments on the basis of nucleotide differences wherein the nucleic acid segments may differ in size, base composition, or both.
2. Summary of the Background
The science of genetics is based on the identification and characterization of mutations, which are changes in DNA (DNA polymorphisms) due to nucleotide substitution, insertion, or deletion. Thus, many techniques have been developed to compare homologous segments of DNA to determine if the segments are identical or if they differ at one or more nucleotides. Practical applications of these techniques include genetic disease diagnoses, forensic techniques, and human genome mapping.
The most definitive method for comparing DNA segments is to determine the complete nucleotide sequence of each segment. Examples of how sequencing has been used to study mutations in human genes are included in the publications of Engelke, et al., Proc. Natl. Acad. Sci. U.S.A. 85:544-548 (1988) and Wong, et al., Nature 330:384-386 (1987). At the present time, it is not practical to use extensive sequencing to compare more than just a few DNA segments, because the effort required to determine, interpret, and compare sequence information is time-consuming.
For genetic mapping purposes, the most commonly used screen for DNA polymorphisms arising from mutation consists of digesting DNA with restriction endonucleases and analyzing the rsulting fragments by means of Southern blots, as described by Botstein, et al.,Am. J. Hum. Genet. 32:314-331 (1980); White, et al., Sci. Am. 258:40-48 (1988). Mutations that affect the recognition sequence of the endonuclease will preclude enzymatic cleavage at that site, thereby alterning the cleavage pattern of that DNA. DNAs are compared by looking for differences in restriction fragment lengths. A major problem with this method (known as restriction fragment length polymorphism mapping or RFLP mapping) is its inability to detect mutations that do not affect cleavage with a restriction endonuclease. Thus, many mutations are missed with this method. One study, by Jeffreys, Cell 18:1-18 (1979), was able to detect only 0.7% of the mutational variants estimated to be present in a 40,000 base pair region of human DNA. Another problem is that the methods used to detect restriction fragment length polymorphisms are very labor intensive, in particular, the techniques involved with Southern blot analysis.
A technique for detecting specific mutations in any segment of DNA is described in Wallace, et al., Nucl. Acids Res. 9:879-894 (1981). It involves hybridizing the DNA to be analyzed (target DNA) with a complementary, labeled oligonucleotide probe. Due to the thermal instability of DNA duplexes containing even a single base pair mismatch, differential melting temperature can be used to distinguish target DNAs that are perfectly complementary to the probe from target DNAs that differ by as little as a single nucleotide. An adaptation of this technique, described by Saiki, et al., U.S. Pat. No. 4,683,194, can be used to detect the presence or absence of a specific restriction site. In Saiki's adaptation, an end-labeled oligonucleotide probe spanning a restriction site is hybridized to the target DNA. The hybridized duplex of DNA is then appropriately incubated with the restriction enzyme for that site. Only paired duplexes between probe and target that reform the restriction site will be cleaved by digestion with the restriction endonuclease. Detection of shortened probe molecules indicates that the specific restriction site is present in the target DNA. In a related technique, described in Landegren, et al., Science 241:1077-1080 (1988), oligonucleotide probes are constructed in pairs such that their junction corresponds to the site on the DNA being analyzed for mutation. These oligonucleotides are then hybridized to the DNA being analyzed. Base pair mismatch between either oligonucleotide and the target DNA at the junction location prevents the efficient joining of the two oligonucleotide probes by DNA ligase. A major problem with these and other oligonucleotide techniques is that the mutation must already be characterized as to type and location in order to synthesize the proper probe. Thus, techniques using oligonucleotide probes can be used to assay for specific, known mutations, but they cannot be used generally to identify previously undetected mutations.
In the technique described in Mundy, U.S. Pat. No. 4,656,127, specific mutations can be detected by first hybridizing a labeled DNA probe to the target nucleic acid in order to form a hybrid in which the 3' end of the probe is positioned adjacent to the specific base being analyzed. Then, a DNA polymerase is used to add a nucleotide analog, such as a thionucleotide, to the probe strand, but only if the analog is complementary to the specific base being analyzed. Finally, the probe-target hybrid is treated with exonuclease III. If the nucleotide analog has been incorporated, the labeled probe is protected from nuclease digestion. Absence of a labeled probe indicates that the analog and the specific base being analyzed were not complementary. As with abovediscussed techniques involving oligonucleotides, this method detects specific mutations, but it cannot be used in a general manner to detect all possible nucleotide differences.
Nucleotide differences between two DNA sequences also can be studied by forming a heteroduplex between the two DNAs of interest. Base pair mismatches will occur within the heteroduplex at points where the sequences differ. A number of methods have been developed to detect such mismatches. Chemical probes for mismatches exist that specifically react with those atoms in the base normally involved in hydrogen bonding, see, e.g., Novack, et al., Proc. Natl. Acad. Sci. U.S.A. 83:586-590 (1986); Cotton, et al., Proc. Natl. Acad. Sci. U.S.A. 85:4397-4401 (1988). These chemically altered sites are susceptible to chemical cleavage, whereas a perfectly paired duplex is not. Problems with this technique include: (i) toxicity of the chemical reagents and (ii) efficiency of much less than 100% for the reactions with unpaired bases. Another approach to mismatch detection is based upon the ability of certain nucleases to recognize and cleave these sites. S.sub.1 nuclease and RNase A have been shown effective in mismatch detection and cleavage, see, e.g., Shenk, et al., Proc. Natl. Acad. Sci. U.S.A. 72:989-993 (1975); Myers, et al., Science 230:1242-1246 (1985). Neither of these enzymes, however, cleaves at all possible mismatched base pairs. There is also considerable background associated with nuclease cleavage at perfectly paired sites in the duplex.
Myers, et al., Nature 313:495-498 (1985), and Fischer, et al., Proc. Natl. Acad. Sci. U.S.A. 80:1579-1583 (1983), have demonstrated that mismatched base pairs within a heteroduplex alter its melting properties with respect to a perfectly paired homoduplex. These altered melting properties can be observed electrophoretically on a gel containing an exponential gradient of denaturant. A serious drawback to this technique is the difficulty of manipulating and processing these gels. Another problem is the inability of this technique to detect uniformly all base pair mismatches along a given heteroduplex. The resolving power of these gels is reduced with increasing GC content of the heteroduplex, and thus mutations in a GC-rich domain are more difficult to detect than mutations in a domain with a lower GC content.
The primer extension process described in Proudfoot, et al., Science 209:1329-1336 (1980), has been widely used to study the structure of RNA and also has been used to characterize DNA, see, e.g. Engelke, et al., Proc. Natl. Acad. Sci. U.S.A. 85:544-548 (1988). This process consists of hybridizing a labeled oligonucleotide primer to a template RNA or DNA and then using a DNA polymerase and deoxynucleoside triphosphates to extend the primer to the 5' end of the template. The labeled primer extension product is then fractionated on the basis of size, usually by electrophoresis through a denaturing polyacrylamide gel. When used to compare homologous DNA segments, this process can detect differences due to nucleotide insertion or deletion. Because size is the sole criterion used to characterize the primer extension product, this method cannot detect differences due to nucleotide substitution.
In order to be useful for a wide variety of applications, a technique to detect nucleotide differences (mutations) in DNA should be simple, fast, and able to detect any nucleotide difference that might occur and, additionally, should not be dependent on the prior characterization of the nucleotide difference. The currently available detection techniques discussed above are deficient in one or more of these areas. Many of the problems associated with these techniques are overcome by the present invention.
The process of the present invention exploits the fact that the incorporation of some nucleotide analogs into DNA causes an incremental shift in mobility when the DNA is subjected to a size fractionation process, such as electrophoresis. Others have noted that nucleotide analogs can cause an electrophoretic mobility shift, see, e.g., Lo, et al., Nucl. Acids Res. 16:8719 (1988); Dattagupta, et al., European Patent Application No. 8602766.2 (published 1986), but it has not been realized, nor is it obvious, that this property of nucleotide analogs can be used as the basis for a process to identify previously undetected mutations.